Effect of 4-trifluoromethyl derivatives of salicylate on nuclear factor κB-dependent transcription in human astrocytoma cells

Eect of 4-tri¯uoromethyl derivatives of salicylate on nuclear factor kB-dependent transcription in human astrocytoma cells 1 Marita Herna Ândez, 2 Alberto Ferna Ândez de Arriba, 2 Manel Merlos, 1 LucõÂa Fuentes, * ,1 Mariano Sa Ânchez Crespo & 3 MarõÂa Luisa Nieto 1 Instituto de BiologõÂa y Gene Âtica Molecular, Consejo Superior de Investigaciones Cientõ®cas, 47005-Valladolid, Spain; 2 Department of Pharmacology, Uriach Research Center, Barcelona, Spain and 3 Instituto de Ciencias del Corazo Ân, Hospital ClõÂnico Universitario, 47005-Valladolid, Spain 1The eect of two derivatives of salicylate, 2-hydroxy-4-tri¯uoromethylbenzoic acid (HTB) and 2- acetoxy-4-tri¯uoromethylbenzoic acid (tri¯usal), on the expression of several proteins displaying proin¯ammatory activities the regulation of which is associated to the transcription factor NF-kB, was assayed in the human astrocytoma cell line 1321N1. 2Tumour necrosis factor-a(TNF-a) activated NF-kB as judged from both the appearance of kB- binding activity in the nuclear extracts, the degradation of IkB proteins in the cell lysates, and the activation of IkB kinases using an immunocomplex kinase assay with glutathione S-transferase (GST)-IkB proteins as substrates. 3HTB up to 3 mMdid not inhibit the nuclear translocation of NK-kB/Rel proteins as judged from electrophoretic mobility-shift assays; however, HTB inhibited the degradation of IkBbwithout signi®cantly aecting the degradation of both IkBaand IkBe. 4In keeping with their inhibitory eect on IkBbdegradation in the cell lysates, both HTB and tri¯usal inhibited the phosphorylation of GST-IkBbelicited by TNF-a, without aecting the phosphorylation of GST-IkBa. 5The eect of both HTB and tri¯usal on kB-dependent trans-activation was studied by assaying the expression of both cyclo-oxygenase-2 (COX-2) and vascular cell adhesion molecule-1 (VCAM-1). HTB and tri¯usal inhibited in a dose-dependent manner the expression of COX-2 and VCAM-1 mRNA and the induction of COX-2 protein at therapeutically relevant concentrations. 6These ®ndings show the complexity of the biochemical mechanisms underlying the activation of NF-kB in the dierent cell types and extend the anti-in¯ammatory eects of HTB and tri¯usal to neural cells. British Journal of Pharmacology (2001) 132, 547 ± 555 Keywords: Adhesion molecules; Alzheimer disease; aspirin; astrocytes; cyclo-oxygenase; gliosis; in¯ammation; nuclear factor kB; tri¯usal; tumour necrosis factor Abbreviations: ALLN, N-acetyl-leucinyl-leucinyl-norleucinal; COX-2, cyclo-oxygenase-2; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; HTB, 2-hydroxy-4- tri¯uoromethylbenzoic acid; IkB, inhibitor of NF-kB activity; IKK, IkB kinase; JNK, c-Jun N-terminal kinase; iNOS, inducible isoform of nitric oxide synthase; NF-kB, nuclear factor kB; NSAIDs, non-steroidal anti-in¯ammatory drugs; PCR, polymerase chain reaction; PPAR, peroxisome proliferator-activated receptors; RT, reverse transcriptase; TNF-a, tumour necrosis factor-a; Tri¯usal, 2-acetoxy-4-tri¯uoromethylbenzoic acid; VCAM-1, vascular cell adhesion molecule-1 Introduction Salicylates are some of the most commonly used antiin¯ammatory agents the action of which has been related to blockade of prostaglandin synthesis via inhibition of cyclooxygenase activity (Vane, 1971; Ferreira et al., 1971), and more recently linked to their ability to inhibit the activation of the transcription factor NF-kB (Kopp & Ghosh, 1994; Grilli et al., 1996; Oeth & Mackman, 1995; Pierce et al., 1996). The association of both pharmacological eects into the same molecule can be a signi®cant advantage for therapeutics, since the inducible isoform of the cyclooxygenase enzyme (COX-2) contains two kB sites in its promoter/enhancer (Appleby et al., 1994), thus explaining that inhibition of kB-binding activity might block the expression of COX-2 and the ensuing production of prostanoids at in¯ammatory sites. The eect of some derivatives of salicylate, namely, the compounds 2-acetoxy- 4-tri¯uoromethylbenzoic acid (trifusal) and 4-tri¯uoromethylbenzoic acid (HTB) (De la Cruz et al., 1992; Rabasseda & Garcia-Rafanell, 1993) has been studied as to their ability to inhibit COX-2 protein expression and prostaglandin E 2 (PGE 2 ) production, on the one hand (Ferna Ândez de Arriba et al., 1999), and kB-dependent trans-activation of some genes such as the chemokine MCP-1 (Alonso et al., 2000), the inducible isoform of nitric oxide syntase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) (Bayo Ânet al., 1999), on the other. NF-kB/Rel plays an important role in gene British Journal of Pharmacology (2001) 132, 547 ± 555 ã 2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00 www.nature.com/bjp *Author for correspondence at: Instituto de Biologõ Âa y Gene Âtica Molecular, Facultad de Medicina, 47005-Valladolid, Spain; E-mail: [email protected] regulation during in¯ammatory reactions in a variety of disease settings, its activation being mediated by a network of kinases leading to the phosphorylation of IkB, which is subsequently degraded by the proteasome, a multicatalytic high molecular weight protease system. The possibility that salicylates might interfere with intracellular kinases has been suggested by the description of the blockade of the activation of the extracellular signal-regulated kinase (ERK) subgroup of mitogen-activated protein kinases in response to TNF-a (Schwenger et al., 1996) and by the involvement of p38 MAP kinase in the inhibitory eect of sodium salicylate on NF-kB activation (Alpert et al., 1999). Interestingly, it has recently been shown that salicylate behaves as a competitive inhibitor of IkB kinase-b(IKKb) by inhibiting the binding of ATP (Yin et al., 1998), which seems to be the main biochemical mechanism explaining its pharmacological eect on NF-kB activation. Interestingly, the biochemical mechanisms leading to the activation of IKK and the sensitivity to pharmacological inhibitors show signi®cant dierences related to the cell type, which might in¯uence the therapeutic applications of antiin¯ammatory drugs. Unlike hemopoietic cells, the proin- ¯ammatory cytokines IL-1aand TNF-ainduce NF-kB- regulated adhesion molecules in astrocytes by a mechanism insensitive to inhibition by the antioxidants pyrrolidine dithiocarbamate and N-acetyl-cysteine (Moynagh et al., 1994). In addition, glucocorticoids, the anti-in¯ammatory eect of which has been related to their ability to inhibit NF- kB activation (Scheinman et al., 1995; Auphan et al., 1995), seem to produce this eect in brain cells by a mechanism dierent from the proposed eect on NF-kB (Bourke & Moynagh, 1999). Moreover, in a study addressing the activation of NF-kB by amiloid Abpeptide, an event likely involved in the Alzheimer's pathogenic pathway, sodium salicylate only inhibited NF-kB activation at concentrations beyond its therapeutic level (410 mM), which might indicate the resistance of NF-kB to salicylate inhibition in glial cells (Dodel et al., 1999). On this basis, we have addressed the eect of both tri¯usal and HTB on the expression of the products of several genes containing kB sites in their promoters, taking into account that the proteins encoded by these genes are involved in in¯ammatory responses in astrocytoma cells. Our data indicate that both tri¯usal and HTB show an inhibitory eect on the phosphorylation and degradation of IkBb, without in¯uencing the outcome of both IkBaand IkBe, as well as a de®nite inhibitory eect on the expression of proin¯ammatory proteins at therapeutically signi®cant concentrations (McNeely & Goa, 1998). These ®ndings extend the inhibitory eect of tri¯usal and HTB on the expression of proin¯ammatory proteins to astrocytoma cells and add further to the complexity of the biochemical mechanisms underlying this eect by disclosing signi®cant dierences between neural cells on the one hand, and endothelial and monocytic cells on the other hand. Methods Cells and reagents 1321N1 astrocytoma cells were cultured in DMEM containing 5% fetal calf serum at 378C in an atmosphere containing 5% CO 2 . Recombinant TNF-awas from Genzyme Diagnostics (Cambridge, MA, U.S.A.). Sodium salicylate was from Fluka Chemika-BioChemika (Buchs, Switzerland). 2- Acetoxy-4-tri¯uoromethylbenzoic acid and 2-hydroxy-4-tri- ¯uoromethylbenzoic acid were from Uriach Laboratories (Barcelona, Spain). Sodium salicylate was dissolved in dimethylsulphoxide and diluted in phosphate-buered saline solution to prepare a 1 Mstock solution. The stock solutions of the remaining drugs were directly made in dimethylsulphoxide. Control cells were treated with the vehicle solution used to convey the drugs. N-acetyl-leucinyl-leucinyl-norleuc- inal (ALLN, calpain I inhibitor, MG-101) was purchased from Sigma (Saint-Louis, MO, U.S.A.). Oligonucleotide primers for the detection of VCAM-1 mRNA by RT ± PCR were designed from human gene sequence (EMBL/Gen Bank AC: M30257), and were 5'-TGTCACTGTAAGCTGCAAG- 3'and 5'-TTCCAGCCTGGTTAATTC-3', corresponding to nucleotides 1090 ± 1108 and 1589 ± 1572 (Osborn et al., 1989). Oligonucleotide primers for the detection of COX-2 mRNA by RT ± PCR were designed from human gene sequence (EMBL/GenBank AC: M90100), and were 5'-TTCAAAT- GAGATTGTGGGAAAATTGCT-3'(sense) and 5'-AGAT- CATCTCTGCCTGAGTATCTT-3'(antisense) corresponding to nucleotides 574 ± 600 and 855 ± 878 of human COX-2 encoding sequence (Hla & Neilson, 1992). 5'-ATCATGTTT- GAGACCTTCAA-3'and 5'-TTGCGCTCAGGAGGAG- CAAT-3', corresponding to nucleotides 405 ± 424 and 1029 ± 1048 were used as primers for the detection of human b-actin mRNA. Rabbit polyclonal antiserum for the detection of human COX-2 was from Cayman Chemical Co., (Ann Arbor, MI, U.S.A.). Antibodies against IkBa, IkBb,IkBe, IKKa, IKKband p38-MAP kinase were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). GST- IkBaand IkBbfusion proteins were a kind gift of Dr Michel Karin, Dept. of Pharmacology, University of California, (San Diego, CA, U.S.A.). Western blot analysis of IkB proteins and COX-2 Cells were washed with ice-cold phosphate-buered saline, and lysed in 0.1 ml of ice-cold extraction buer containing 10 mMHEPES, 2 mMEGTA, 10% glycerol, 10 mgml 71 leupeptin, and 1 mMphenylmethylsulphonyl ¯uoride, pH 7.4. Cell lysate was mixed with Laemmli's buer and boiled for 5 min. The amount of protein in each sample was assayed using the Bradford reagent and equal amounts of protein were loaded on each lane of a 10% SDS ± PAGE gel. Proteins were transferred to nitrocellulose membranes using a semi-dry transfer module. The membranes were blocked with BSA for 2 h, washed with Tris-buered saline containing Tween 20, and used for immunoblotting using anti-IkBor anti-COX-2 antibodies followed by donkey anti-rabbit IgG- horseradish peroxidase antibody. Detection was performed using the Amersham ECL system. Assay of IkB kinase activity Anti-IKKaand anti-IKKbantibodies were used for the incubation with the cell lysates, followed by the addition of GammaBind G-Sepharose to trap the antibody/kinase complex according to standard protocols (Herna Ândez et al., 1997; 1999). The kinase reaction was carried out with 500 ng British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al548 of either GST-IkBaor IkBbfusion protein as substrate and 20 mMATP and 5 mCi [g- 32 P]-ATP in a volume of 30 ml. The reaction was diluted in buer and centrifuged to discard supernatant and then boiled in Laemmli SDS sample buer and DTT. Phosphorylated GST-IkB was resolved by 10% SDS ± PAGE and detected with the Bio-Rad Molecular Imager FX system. c-Jun N-terminal kinase was assayed using GST-c-Jun fusion protein as the substrate. Phosphorylated GST-c-Jun was resolved by 10% SDS ± PAGE and immunodetected by Western blot using rabbit phosphospeci®c c-Jun (Ser 63 ) antibody (Herna Ândez et al., 1998). The activation of p38-MAP kinase was assessed by immunoprecipitation of the kinase with anti-p38-MAP kinase antibody, and assay of the extent of tyrosine phosphorylation with antiphosphotyrosine speci®c antibody. Electrophoretic mobility shift assay This was carried out according to previously described protocols (Bayo Ânet al., 1997). Brie¯y, 1321N1 cells were washed with ice-cold hypotonic lysis buer, and allowed to swell on ice for 10 min. Unbroken cells were eliminated by centrifugation and the nuclei were collected by centrifugation at 15,0006gfor 1 min in a microcentrifuge. 22-mer doublestranded oligonucleotide probes containing NF-kB were end-labelled with [g- 32 P]-ATP using T4 polynucleotide kinase. The kB sequence used was, sense 5'-AGTTCAGGG- GAATTTCCCAGGC-3'and the complement 5'- GCCTGGGAAATTCCCCTGAACT-3'. Nucleoprotein-oli- gonucleotide complexes were resolved by electrophoresis in a 4% nondenaturing polyacrylamide gel in Tris borate/ EDTA electrophoresis buer at 175 V for 3 h at 48C. The speci®city of the DNA-protein complex was con®rmed by competition with a 100 fold molar excess of unlabelled nucleotide containing the consensus sequence. Synthesis of first strand cDNA and PCR of VCAM-1 and COX-2 Total cellular RNA was extracted from culture plates according to the guanidium isothiocyanate method (Chomczynski & Sacchi, 1987). cDNA ®rst strand was synthesized from total RNA by reverse transcription reaction. The reaction mixture containing 0.2 mg ml 71 total RNA, 2.5 ml H 2 O, 20 u of RNasin ribonuclease inhibitor, 4 ml buer 56, 2ml DTT 0.1 M,4ml dNTP 2.5 mM,1ml hexanucleotide 0.1 mM, and 200 u of Moloney-murine leukemia virus reverse transcriptase. The cDNA was ampli®ed by PCR in a reaction mixture containing 2 ml of DNA template, 10 mlH 2 O, 2.5 ml buer 106, 0.75 ml MgCl 2 50 mM, 1.0 ml dNTP 2.5 mM, 1.25 ml of each sense and antisense primers and 0.25 mlof Taq DNA polymerase 5 u ml 71 . The ampli®cation pro®le included: one cycle of initial denaturation at 948C for 5 min, 30 cycles of denaturation at 948C for 30 s, primer annealing at 598C for 30 s, and extension at 728C for 1 min; one cycle of ®nal extension at 728C for 7 min. The relative amounts of each ampli®ed cDNA were determined by measuring the density of the bands stained by ethidium bromide using the Gel Doc video gel documentation system and the Molecular Analyst software from Bio-Rad Laboratories (Hercules, CA, U.S.A.). The expression of b-actin was used as control for the assay of a constitutively expressed gene. Statistical analysis Results are expressed as mean+s.e.mean. For comparison of two groups of samples normally distributed, Student's twotailed t-test was used. Results HTB does not produce inhibition of NF-kB activity in EMSA assays, but decreases the degradation of IkBb Stimulation of 1321N1 cells with concentrations of TNF-aas low as 25 u ml 71 , induces a rapid and long-lasting activation of NF-kB (at least 6 h), as judged from the presence of kB- binding protein containing p65 and p50 proteins of the Rel family in the nuclear extract (Herna Ândez et al., 1999). Attempts to ascertain the possible inhibitory eect of HTB on the activation of NF-kB was ®rst addressed by looking at the eect of this compound on the nuclear translocation of NF-kB/Rel proteins. As shown in Figure 1A, concentrations of both sodium salicylate and HTB up to 3 mM, did not Figure 1 Electrophoretic mobility shift assays showing kB-binding activity in nuclear extracts from 1321N1 cells stimulated with TNF-a. Cells were stimulated with 100 u ml 71 TNF-afor 15 min in the presence of the additions indicated and at the end of this time, cell lysates were processed for the assay of kB-binding activity in the nuclear extracts. The lane marked competitor indicates that the binding reaction was carried out with a 100 fold molar excess of unlabelled nucleotide containing the consensus sequence. This is an experiment of four identical ones (A). In the experiment shown in (B), nuclear extracts from cells stimulated with 100 u ml 71 TNF-a for 15 min in the presence of 3 mMHTB were incubated for 15 min at 48C with a 1 : 40 dilution of polyclonal rabbit antibodies prior to the addition of the 32 P-labelled oligonucleotide probe. The proteinoligonucleotide complexes supershifted by the antibodies are noted by an arrow. British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al 549 in¯uence nuclear translocation of proteins displaying kB- binding activity, thus agreeing with previous observations in astrocytoma cells which suggest insensitivity of NF-kB activation to compounds active on other cell types, for example, pyrrolidine dithiocarbamate and N-acetyl-cysteine (Bourke & Moynagh, 1999; Herna Ândez et al., 1999; Dodel et al., 1999), or alternatively, that the eect of these drugs on NF-kB activation is exerted at de®ned molecular steps not disclosed by the EMSA approach. To obtain further insight into these mechanisms, attempts to disclose the composition of the kB-binding complexes with speci®c antibodies were carried out. As shown in Figure 1B, anti-p50 antibody produced a partial shift of the protein-oligonucleotide complexes, whereas anti p65 antibody produced a complete supershift, thus indicating that at least at the time of the experiments, the complexes showed an overall composition similar to that disclosed in the absence of HTB (Herna Ândez et al., 1999). TNF-ainduced a marked degradation of IkB proteins showing distinct kinetics for the dierent proteins (Figure 2A). IkBashowed the earliest degradation, undetectable levels being found as soon as 5 min after addition of the stimulus and reaching pre-stimulation levels after 60 min. IkBbdecreased after 10 min and was almost undetectable for at least 6 h after challenge (Figure 3). IkBedecreased after 15 min and reappeared after 2 h. These data suggests that IkBa-dependent release of NF-kB may not fully account for the long-lasting induction of IkB-binding activity involved in the regulation of NF-kB-dependent transcriptional regulation, thus agreeing with ®ndings in U937 cells where TNF-a and IL-1bproduce a similar activation of kB-binding activity, whereas TNF-aproduces a complete loss of IkBaand IL-1b only produces a 40% reduction (Nasuhara et al., 1999). Interestingly, preincubation with HTB at concentrations 0.5 ± 1m Minhibited the degradation of IkBb(Figure 2B) at a similar extent to that produced by 100 mMALLN (Figure 2C), i.e., a proteasome inhibitor which blocks the nuclear translocation of NF-kB and aects the expression of adhesion molecules and COX-2 (Read et al., 1995; Gallois et al., 1998). However, the levels of both IkBa(Figure 2B) and IkBe(not shown) were not in¯uenced. Quantitation by densitometric scanning of the IkBbblots from samples obtained 15 min after the addition of TNF-ashowed a 39+7% inhibition for 0.5 mMHTB, and a 61+9% inhibition at 1 mM(n=5, P50.05), thus con®rming the anticipated paradigm that using only the EMSA approach, inhibitions of NF-kB activation exerted by agents only acting on some elements of the IkB family might be overlooked. Noteworthy, the viability of the cells was not aected by the concentrations of drugs used, which in fact were selected on the basis of the pharmacological concentrations of these agents that can be obtained in the serum of patients (McNeely & Goa, 1998). HTB and triflusal inhibit the phosphorylation of IkBbby IKK Since it has been described a selective inhibitory eect of sodium salicylate on IKKbfrom both COS and HeLa cells (Yin et al., 1998), the eect of tri¯usal and HTB on IKK activity was assessed. As shown in Figure 4, TNF-ainduced a rapid and transient activation of IKK in 1321N1 cells, as judged from the presence of IKK activity in immunoprecipitates obtained with each of the antibodies, thus agreeing with current views stressing the association of both IKK in the signalsome. Maximal activity was observed at 5 ± 10 min, and decreased to reach prestimulation values by 15 ± 30 min. Figure 2 Immunodetection of IkB proteins in 1321N1 cells stimulated with 100 u ml 71 TNF-a. Cells were stimulated with TNF-afor the times indicated and at the end of these periods the cell lysate was used for SDS ± PAGE and immunodetection of IkBa, IkBb, and IkBe(A). The same membrane was used for the sequential immunodetection of IkBaand IkBbwith their cognate antibodies, whereas IkBewas immunoblotted in a separate membrane. (B) Represents the eect of dierent concentrations of HTB on the degradation of IkBb. (C) Shows the eect of dierent concentrations of ALLN. The assay of IkBbin (B) and (C) was conducted on cell lysates collected 15 min after the addition of TNF-a. Figure 3 Resynthesis of IkBaand IkBein 1321N1 cells after stimulation with TNF7a. The conditions of the assay were as those in Figure 2, but the cell lysates were collected at the time indicated to assess the resynthesis at later times. British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al550 Under the assay conditions, no dierences of enzyme activity towards each of the substrates were observed, which agrees with the aforementioned ability of TNF-ato induce degradation of both IkBaand IkBb. The relative amount of each IKK present in 1321N1 cells was addressed by immunodetection in the cell lysates and compared to that detected in THP-1 cells. As shown in Figure 4C, THP-1 cells showed a signi®cantly higher amount of IKKbthan 1321N1 cells. Preincubation of 1321N1 cells with both HTB and tri¯usal at the concentration of 1 mMprior to the addition of TNF-adiminished the IKK activity associated to the immunoprecipates when GST-IkBbwas used as a substrate, whereas no inhibition was observed when the assay was conducted with GST-IkBa(Figure 5), thus agreeing with the diminished degradation of IkBbin whole cells incubated with HTB. Attempts to address the eect of HTB on other kinases that are activated by TNF-afocused on JNK, since unlike the ERK module of MAP kinases, activation of the stress module of MAP kinases, i.e., JNK and at lower extent p38- MAP kinase, is a hallmark of the response to TNF-ain astrocytoma cells (Herna Ândez et al., 1999). As shown in Figure 6, HTB at the highest concentration used throughout these experiments did not signi®cantly aect JNK activity nor the tyrosine phosphorylation of p38-MAP kinase, thus suggesting that the eects herein reported can not be explained by inhibition of the stress module of MAP kinases. HTB and triflusal inhibit the expression of COX-2 and VCAM-1 induced by TNF-ain astrocytoma cells Attempts to address whether the aforementioned eects of tri¯usal and HTB on IkBbphosphorylation and degradation might have consequences on the regulation of IkB-regulated genes, experiments were carried out to assess the eect of these compounds on VCAM-1 and COX-2 mRNA as prototypic inducible genes expressed in astrocytoma cells the expression of which has been found to be inhibited in other cell types (Bayo Ânet al., 1999; Ferna Ândez de Arriba et al., 1999). As shown in Figure 7A, incubation with HTB in the range 1 ± 3 mM, induced a signi®cant inhibition of the expression of COX-2 mRNA induced by TNF-aas judged from RT ± PCR reactions, whereas sodium salicylate at the same doses failed to produce inhibitions higher than 20%. This was accompanied by a reduction of COX-2 protein expression as judged from Western blot assays carried out under identical conditions. As shown in Figure 7B, 1 mM HTB produced a 58+12% inhibition of COX-2 expression induced by TNF-a, whereas the inhibition with 3 mMHTB was 92+6%. The expression of VCAM-1 mRNA, which shows a long-lasting induction maintained for at least 6 h after stimulation with TNF-a(Figure 8A), was also inhibited by HTB in the range 1 ± 5 mM(Figure 8B). Discussion The initial description of the inhibitory eect of aspirin and sodium salicylate on the activation of the transcription factor NF-kB (Kopp & Ghosh, 1994; Weber et al., 1995; Grilli et al., 1996; Pierce et al., 1996), has opened new avenues to explain the therapeutic properties of salicylate derivatives, and has been followed by the description of analogous properties of some derivatives including sulfasalazine (Wahl et al., 1998) and 2-hydroxy-4-tri¯uoromethylbenzoic acid (Bayo Ânet al., 1999; Ferna Ândez de Arriba et al., 1999), and by the description of new mechanisms of action for other nonsteroidal anti-in¯ammatory agents, for instance sulindac (Yamamoto et al., 1999), pari passu with the unveiling of Figure 4 Activation of IKK in 1321N1 cells stimulated with TNF-a. 1311N1 cells were incubated in the presence of 100 u ml 71 of TNF-afor the times indicated. Cell lysates were collected for immunoprecipitation of the signalsome with anti-IKK and the assay of IKK activity with the immunoprecipitated kinase and either GST-IkBaor GST-IkBb. Cells incubated for 120 min in the absence of TNF-awere also included as controls. The experiment shown in (A) corresponds to an experiment carried out with anti-IKKa, whereas the experiment shown in (B) was carried out using anti-IKKbantibody for the immunoprecipitation. (C) Compares the amount of both IKKaand IKKbin cell lysates from 1321N1 and THP-1 cells. P-, phosphorylated. British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al 551 the mechanism of activation of NF-kB. Namely, the role for salicylate as a speci®c inhibitor of IKKb(Yin et al., 1998), and the description of cell-speci®c mechanisms of activation of NF-kB, which might explain distinct properties of pharmacological agents on some cell types. Taking these reports into account, we have extended previous studies in human umbilical vein endothelial cells (Bayo Ânet al., 1999) and THP-1 monocytic cells (Alonso et al., 2000) to human astrocytoma cells in view of the role for astrocytes in in¯ammatory and degenerative neural diseases, and the reported evidence of unique pharmacological resistance of these cells to inhibitors of the activation of NF-kB that are operative in other cell types (Moynagh et al., 1994; Bourke & Moynagh, 1999). Our data agree with these reports (Moynagh et al., 1994; Bourke & Moynagh, 1999) by showing that unlike umbilical Figure 5 Eect of salicylate, HTB and tri¯usal on IKK activity. 1321N1 cells were stimulated with 100 u ml 71 of TNF-afor 5 min in the presence of the indicated additions. At the end of this period, cell lysates were used for immunoprecipitation with anti-IKKaand anti- IKKbantibodies and used for in vitro kinase assay with both GST- IkBaand GST-IkBbas substrates (A). The histogram (B) shows the quantitation of ®ve independent experiments carried out with the immunoprecipitates obtained using both anti-IKKa(open bars) and anti-IKKb(striped bars) antibodies and GST-IkBbas substrate. *P50.05. Figure 6 Eect of HTB on the activation of JNK and p38-MAP kinase tyrosine phosphorylation. 1321N1 cells were incubated with 100 u ml 71 TNF-afor 15 min, in the presence and absence of 3 mM HTB. At the end of this period cell lysates were collected for the assay of P-GST-c-Jun phosphorylation with phosphospeci®c anti-c- Jun antibody. This is a representative experiment of three identical ones (upper panel). The lower panel shows a typical experiment carried out under identical conditions, in which cell lysates were used for immunoprecipitation with anti-p38-antibody, SDS ± PAGE separation of the immunoprecipitate, and blotting with antiphosphotyrosine antibody. P-Y, phosphotyrosine. Figure 7 Eect of the incubation with sodium salicylate and HTB on the expression of COX-2 mRNA in cells incubated for 6 h in the presence of 100 u ml 71 TNF-a(A). This is a representative experiment of ®ve identical ones. Eect of salicylate and HTB on the expression of COX-2 protein induced by TNF-a. Cells were incubated with 100 u ml 71 TNF-afor 4 h and then the cell lysates were collected for the immunodetection of COX-2 protein (B). The histogram in panel (B) shows mean+s.e.mean of the densitometric scanning of four independent experiments. British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al552 vein endothelial cells and THP-1 cells, HTB is not able to block the nuclear translocation of NF-kB, as judged from EMSA assays, at pharmacologically relevant concentrations. However, it blocked the degradation of IkBbin cell lysates at a similar extent to that found with the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal, which in contrast to the antioxidants pyrrolidine dithiocarbamate and N-acetyl- cysteine was active in 1321N1 cells. These data stress the complexity of the NF-kB/Rel system and the requirement for dierent experimental approaches addressing the involvement of the dierent elements of the family before ruling out its involvement in some settings. For instance, NF-kB/Rel p50 homodimers, which lack kB-trans-activating activity can be present in nuclear extracts from RBL5 cells incubated for several hours in the presence of sulfasalazine under conditions where other NF-kB/Rel heterodimers are blocked (Liptay et al., 1999). Our data agree with the reported eect of salicylate as an almost irreversible inhibitor of IKK the eect of which is still observed in the cell-free system (Yin et al., 1998). However, our data do not allow to state whether the eect of HTB is exerted on either IKKaor IKKbsince both isoforms are associated to the proteasome and are immunoprecipitated in our system irrespective of the speci®city of the antibody used. Attempts to characterize the isoforms expressed in 1321N1 cells have disclosed a lower amount of IKKbthan in THP-1 monocytic cells. This seems of interest, since activation of THP-1 cells by lipopolysaccharide and TNF-aproduces dierent patterns of activation of IKKs for each stimulus. In fact, lipopolysaccharide predominantly produces a long-lasting activation of IKKb activity, whereas TNF-aproduces a rapid and short-lived predominant activation of IKKa(O'Connell et al., 1998; Fischer et al., 1999). On this basis, the increased expression of IKKbin THP-1 cells and monocytes might explain a privileged functional role for this IKK in these cells, and might also favour the chance of obtaining selective immunoprecipitation of each IKK by disrupting the signalsome by adding Triton X-100 to the immunoprecipitation buer, as described by Fischer et al. (1999). It should be noted, however, that this approach was unsuccessful in our hands. The functional signi®cance of IkBbdegradation should also be discussed on the basis of previous studies in 1321N1 cells. For instance, glucocorticoids inhibit the expression of IL-8, VCAM-1 and ICAM-1 in the absence of inhibition of both nuclear translocation of NF-kB proteins and degradation of IkBa(Bourke & Moynagh, 1999). However, since the degradation of IkBbwas not assessed in this report, the chance that glucocorticoids do aect the phosphorylation and degradation of IkBbcan not be ruled out. Alternatively, the eect of some anti-in¯ammatory agents previously described as inhibitors of NF-kB activation should be explained on the basis of other pharmacological properties. In fact, it has been reported using sensitive transfection experiments, that the expression of other NF-kB regulated genes (e.g. P-selectin) is inhibited by sodium salicylate without a detectable requirement for an intact kB element in the P-selectin gene (Xia et al., 1998). In addition, inhibition by both aspirin and sodium salicylate of nitric oxide production exerted at a translational step have also been reported in hepatocytes (Sakitani et al., 1997), thus agreeing with earlier suggestions that a portion of the eect of salicylates on iNOS induction could be explained by mechanisms other than inhibition of kB-dependent transactivation (Farivar & Brecher, 1996). These cautions seem particularly adequate for the eects observed on COX-2, since in addition to kB sites a role for peroxisome proliferator-activated receptors (PPAR) in the trans-activa- tion of COX-2 has been reported, thus indicating that PPAR activators, e.g. non-steroidal anti-in¯ammatory drugs (NSAIDs), might under some conditions enhance the expression of COX-2 in epithelial cells (Meade et al., 1999). However, the net eect of PPAR agonists on COX-2 expression induced by pro-in¯ammatory cytokines is inhibitory, since the prototypical compounds sulindac disul®de and ¯ufenamic acid only enhance COX-2 expression at concentrations below 200 mM, whereas via their inhibitory eect on NF-kB activation, they inhibit the induction elicited by proin¯ammatory cytokines at concentrations above 200 mM (Paik et al., 2000). Irrespective of the biochemical mechanism that might explain the distinct eect of HTB on the phosphorylation and degradation of IkBb, our results indicate a signi®cant eect of HTB on kB-dependent trans-activation by showing a decreased expression of mRNA of both COX-2 and VCAM- 1, both molecules being of relevance for the functional involvement of astrocytes in immunoin¯ammatory conditions. However, as the inhibition of COX-2 protein expression was bigger than the inhibition of its mRNA expression, an additional post-translational eect of HTB contributing to the down regulation of COX-2 seems likely (Mitchell et al., 1997). These ®ndings may have implications for the therapeutic applications of tri¯uoromethyl derivatives of salicylates in view of the prominent eects of HTB on both VCAM-1 and COX-2 expression in neural cells, and the pharmacokinetics of HTB which shows a t 1/2 of 35 h after repeated doses in humans (McNeely & Goa, 1998), as compared to a half-life of 2.4 h of salicylate at therapeutic doses (Insel, 1991). Figure 8 Eect of HTB on VCAM-1 expression. Cells were incubated with 100 u ml 71 TNF-afor the times indicated and then processed for the detection of VCAM-1 mRNA by RT ± PCR (A). (B) Shows an experiment of three carried out with cells stimulated 4 h with TNF-ain the presence of dierent concentrations of salicylate and HTB. The expression of b-actin is shown for comparison. British Journal of Pharmacology vol 132 (2) Triflusal inhibition of NF-kBM. HernaÂndez et al 553 Dr Michel Karin is thanked for the gift of GST-IkBaand IkBb fusion proteins. 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